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Amoebae in Moisture- Damaged Buildings

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Terhi Yli-Pirilä RESEARCH Amoebae in Moisture- Damaged Buildings 13 Terhi Yli-Pirilä AMOEBAE IN MOISTURE-DAMAGED BUILDINGS ACADEMIC DISSERTATION To be presented with the permission of the Faculty of Natural
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Terhi Yli-Pirilä RESEARCH Amoebae in Moisture- Damaged Buildings 13 Terhi Yli-Pirilä AMOEBAE IN MOISTURE-DAMAGED BUILDINGS ACADEMIC DISSERTATION To be presented with the permission of the Faculty of Natural and Environmental Sciences, University of Kuopio, for public examination in ML3 auditorium, Medistudia Building, on 8 th May 2009, at 12 o clock noon. National Institute for Health and Welfare, Helsinki, Finland and Faculty of Environmental Sciences, University of Kuopio, Finland Kuopio 2009 Publications of the National Institute for Health and Welfare THL. Research 13. Copyright Terhi Yli-Pirilä and National Institute for Health and Welfare ISBN (printed) ISSN (printed) ISBN (pdf) ISSN (pdf) Kannen kuva - cover graphic: Gummerus printing Jyväskylä, Finland 2009 Supervised by Research Professor Aino Nevalainen, Ph.D. National Institute for Health and Welfare, Kuopio, Finland Professor Maija-Riitta Hirvonen, Ph.D. National Institute for Health and Welfare, Kuopio, Finland Department of Environmental Science, University of Kuopio, Finland Adjunct Professor Markku Seuri, Ph.D., M.D. Metso Occupational Health, Jyväskylä, Finland Reviewed by Professor Emeritus Stuart S. Bamforth, Ph.D. University of Tulane, New Orleans, Louisiana, USA Assoc. Professor Rafal L. Górny, Ph.D. Institute of Occupational Medicine and Environmental Health, Sosnowiec, Poland Opponent Adjunct Professor Auli Rantio-Lehtimäki, Ph.D. Turku Centre for Environmental Research, University of Turku, Turku, Finland Ellille ja Pasille Terhi Yli-Pirilä, Amoebae in Moisture-Damaged Buildings National Institute for Health and Welfare, Research Pages. Helsinki, Finland ISBN (printed), (pdf) ABSTRACT Moisture damage in buildings and consequent microbial growth are associated with adverse health effects suffered by the occupants. Although the association is well documented epidemiologically, the exact causative agents for the health effects are not usually known. Even though the microbial network growing on moisturedamaged building materials is a complex ecosystem consisting of many types of organisms including bacteria, yeasts, fungi, protozoa, and mites, exposure has mainly been described in terms of fungal and bacterial diversity and quantity. It is important not to overlook the other possible organisms growing on these materials to better understand the link between the exposure and the symptoms. In this thesis, the occurrence and role of amoebae in moisture damage is elucidated. First, the prevalence of amoebae in moisture-damaged buildings was estimated by screening 124 building material samples. Then amoebal survival on moist building materials was studied by inoculating samples of building materials with Acanthamoeba polyphaga and incubating those in 100 % relative humidity for 0-56 days. Thirdly, the effects of amoebae on other microbes commonly found in moisture-damaged buildings were assessed by co-cultivating three bacterial (Streptomyces californicus, Bacillus cereus, and Pseudomonas fluorescens) and three fungal strains (Stachybotrys chartarum, Aspergillus versicolor, and Penicillium spinulosum) together with A. polyphaga and also individually for up to 28 days. Their growth was measured at different times during the incubation. Finally, the effects of this co-culture on the cytotoxic and proinflammatory potential of the microbes were studied by exposing RAW264.7 mouse macrophages to graded doses of co-cultured and individually grown fungi, bacteria and amoebae. Amoebae were found in 22 % of the samples and they often were detected at the same locations as indicator microbes of moisture damage, e.g. with the bacterium Streptomyces, and with the fungi Acremonium, Trichoderma, Chaetomium, and Aspergillus versicolor. In the inoculation tests, A. polyphaga amoebae survived throughout the two-month experiment on samples of mineral insulation, old pine plank, birch plank and gypsum board, often even without nutrient supplementation. All materials with the exception of fresh pine plank, supported amoebal survival at least temporarily. Furthermore, co-cultivation with amoebae significantly increased the growth of all bacteria studied, whereas with fungi, only a modest increase in the growth was observed. Co-culturing also affected the toxicity and proinflammatory potential of two of the studied strains: the ability of P. spinulosum and S. californicus to induce the production of inflammatory mediators - nitric oxide, TNFα and IL-6 - in RAW264.7 macrophages was increased manifold. In addition, their cytotoxicity was somewhat increased after incubation with amoebae. The results of this study show that amoebae are members of the microbial network present in moisture-damaged building materials. The interaction with amoebae could lead to alterations in the properties of the other microbes present in the waterdamaged structures. Amoebae may increase the growth of other microbes present, and render the microbes more cytotoxic. Thus, amoebae may indirectly modify the health effects associated with moisture-damaged buildings. However, more evidence from both empirical and epidemiological studies is needed before the role of amoebae as exposing agents in moisture-damaged buildings is fully understood. Keywords: Amoebae, Acanthamoeba polyphaga, bacteria, fungi, moisture damage, buildings, building materials, co-culture, cytokines, NO Terhi Yli-Pirilä. Amoebae in Moisture-Damaged Buildings. Terveyden ja hyvinvoinnin laitos, Research 13. Helsinki sivua. ISBN (painettu), (pdf) TIIVISTELMÄ Rakennusten kosteusvauriot ja mikrobikasvusto ovat yhteydessä rakennusten käyttäjien kokemiin terveyshaittoihin. Vaikka tämä yhteys on osoitettu epidemiologisesti, tarkkoja terveysvaikutuksien aiheuttajia ei tunneta. Kosteusvaurioissa esiintyvä mikrobikasvusto on monimutkainen ekologinen kokonaisuus, jossa on mukana bakteereita, homesieniä, hiivoja, alkueläimiä ja punkkeja. Altistusta kuvataan tavallisesti mittaamalla homesienten ja joskus bakteereiden lajikirjoa ja pitoisuutta. Jotta terveysvaikutuksen ja kosteusvaurioituneessa rakennuksessa esiintyvän altistuksen välistä yhteyttä voitaisiin selventää, on tärkeää tutkia myös muita kosteusvaurioissa esiintyviä eliöitä. Tässä väitöskirjatyössä tutkittiin ameboiden esiintymistä ja vaikutuksia kosteus- ja homevaurioituneissa materiaaleissa. Ameboiden yleisyyttä kosteusvaurioituneissa rakennuksissa selvitettiin viljelemällä amebat 124 rakennusmateriaalinäytteestä. Amebojen selviytymistä eri rakennusmateriaaleilla seurattiin ymppäämällä Acanthamoeba polyphaga -amebaa rakennusmateriaalinäytteisiin ja inkuboimalla näitä 100% suhteellisessa kosteudessa 0-56 vrk ajan. Tutkimuksen kolmannessa osassa mitattiin amebojen vaikutuksia muihin kosteusvauriomikrobeihin kasvattamalla kosteusvauriorakennuksista eristettyjä kolmea bakteerikantaa (Streptomyces californicus, Bacillus cereus ja Pseudomonas fluorescens) ja kolmea homesienikantaa (Stachybotrys chartarum, Aspergillus versicolor, ja Penicillium spinulosum) erikseen ja yhdessä A. polyphaga -amebakannan kanssa 0-28 vrk ajan. Homesienten, bakteerien ja amebojen kokonaispitoisuudet ja elinkykyisten itiöiden/solujen pitoisuudet määritettiin useissa aikapisteissä inkuboinnin aikana. Lopuksi selvitettiin myös amebojen vaikutuksia näiden kantojen toksisuuteen ja kykyyn aiheuttaa tulehdusvasteita altistamalla hiiren makrofageja (RAW264.7) erisuuruisille annoksille ko. mikrobien itiöitä/soluja. Ameboja löydettiin 22 % kosteusvaurioituneista rakennuksista otetuista näytteistä, ja ne esiintyivät usein yhdessä kosteusvaurioiden indikaattorimikrobien kanssa, kuten Streptomyces-bakteereiden ja Acremonium, Trichoderma, Chaetomium, ja Aspergillus versicolor -homesienten kanssa. Kasvatuskokeissa havaittiin, että A. polyphaga selvisi elinkykyisenä koko kahden kuukauden inkuboinnin ajan mineraalivillalla, harmaantuneella mäntylankulla, koivulankulla ja kipsilevyllä jopa ilman lisättyä ravintoa. Amebat selvisivät myös muilla materiaaleilla tuoretta mäntyä lukuun ottamatta ainakin hetkellisesti. Ameban vaikutuksia muihin mikrobeihin selvitettäessä havaittiin, että yhteiskasvatus ameban kanssa lisäsi merkitsevästi kaikkien bakteereiden kasvua ja elinkykyä. Homesienille vaikutus oli vähäisempi. Yhteiskasvatus myös lisäsi kahden tutkitun mikrobikannan toksisuutta ja kykyä aiheuttaa tulehdusvasteita; P. spinulosum -homesienen ja S. californicus - bakteerin kyky indusoida tulehdusvälittäjäaineiden (typpioksidi, TNF α ja IL -6) tuotantoa RAW makrofageissa moninkertaistui. Myös sytotoksisuus lisääntyi jonkin verran. Tämä tutkimus osoittaa, että amebat ovat osa kosteusvaurioituneissa rakennuksissa esiintyvää mikrobiverkostoa. Vuorovaikutus amebojen kanssa voi muuntaa toisten kosteusvaurioissa kasvavien mikrobien ominaisuuksia siten, että amebat voivat lisätä niiden kasvua ja elinkykyä, sekä vaikuttaa näiden tulehdusvasteita aiheuttaviin ominaisuuksiin. On siis mahdollista, että amebat voivat olla epäsuorasti osallisia kosteusvauriorakennuksiin yhdistetyissä terveyshaitoissa. Tarvitaan kuitenkin lisää sekä kokeellista että epidemiologista tutkimusta, jotta amebojen osuus kosteusvaurioituneissa rakennuksissa tapahtuvassa altistumisessa selviäisi. Avainsanat: Amebat, Acanthamoeba polyphaga, bakteerit, sienet, kosteusvaurio, rakennus, yhteiskasvatus, sytokiinit, NO CONTENTS Abbreviations List of original publications Introduction Review of the literature MOISTURE-DAMAGE IN BUILDINGS The exposing agents and health effects associated with moisturedamaged buildings Microbial growth in moisture-damaged buildings Proinflammatory and cytotoxic responses induced in vitro by microbes from moisture-damaged buildings AMOEBAE Free-living amoebae Amoebae and bacteria Amoebae and intracellular microbes other than bacteria Identification methods of environmental amoebae Aims of the study Materials and methods OVERALL STUDY DESIGN THE OCCURRENCE OF AMOEBAE IN BUILDING MATERIAL SAMPLES FROM MOISTURE-DAMAGED BUILDINGS (I) Sampling of building materials (I) Detection and quantification of amoebae from building material samples (I) Detection and identification of fungi and bacteria from the building material samples (I) THE SURVIVAL OF AMOEBAE ON BUILDING MATERIALS (II) THE EFFECTS OF CO-CULTIVATION OF FUNGI AND BACTERIA WITH AMOEBAE ON THEIR GROWTH, VIABILITY, AND IMMUNOTOXIC POTENTIAL (III, IV) Upkeep and preparation of microbial cultures In vitro studies (IV) STATISTICAL ANALYSES... 50 5 Results THE OCCURRENCE OF AMOEBAE IN BUILDING MATERIAL SAMPLES FROM MOISTURE-DAMAGED BUILDINGS (I) SURVIVAL OF AMOEBAE ON DIFFERENT BUILDING MATERIALS (II) THE EFFECTS OF CO-CULTIVATION OF AMOEBAE WITH FUNGI OR BACTERIA ON THEIR GROWTH AND VIABILITY (III) THE EFFECTS OF CO-CULTIVATION WITH AMOEBAE ON CYTOTOXICITY AND PROINFLAMMATORY POTENTIAL OF MICROBES (IV) Discussion THE ROLE OF AMOEBAE AS MEMBERS OF MICROBIAL NETWORK IN MOISTURE DAMAGE AMOEBAL SURVIVAL IN MOISTURE-DAMAGED BUILDINGS THE EFFECTS OF AMOEBAE ON MICROBIAL EXPOSURE IN MOISTURE- DAMAGED BUILDINGS METHODOLOGICAL CONSIDERATIONS Culturing methods of fungi and bacteria (I, III, IV) Detection of amoebae on building material samples (I, II) Immunotoxicological analyses (IV) IMPLICATIONS FOR FUTURE PRACTICE AND RESEARCH Conclusions Acknowledgements References... 72 ABBREVIATIONS ANOVA DG-18 ELISA Free-living amoebae (FLA) Hagem HBSS IL-6 Analysis of variance Dichloran Glycerol agar, a growth medium for fungi with lower moisture requirements Enzyme-linked immunosorbent assay Environmental amoebae that survive and grow without a host organism Rose Bengal malt extract agar, a colony size restrictive growth medium for hydrophilic fungi Hank s Balanced Salt Solution, a cell substrate solution Interleukin 6 cytokine, an inflammatory marker 2 % MEA 2 % Malt Extract agar, a growth medium for hydrophilic fungi MTT NNA NNA-method NO PYG RAW264.7 TNFα TYG 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, reagent used in cytotoxicity test Non-nutritive agar, an amoebal growth medium A method for detecting amoebae with nonnutritive agar plates and roughly estimating their abundance Nitric oxide, an inflammatory marker Peptone Yeast Glucose broth, an amoebal growth medium A mouse macrophage cell line Tumor Necrosis Factor alpha cytokine, an inflammatory marker Tryptone Yeast Glucose agar, a bacterial growth medium 11 LIST OF ORIGINAL PUBLICATIONS This thesis is based on the following original articles referred to in the text by their Roman numerals: I Yli-Pirilä T., Kusnetsov J., Haatainen S., Hänninen M., Jalava P., Reiman M., Seuri M., Hirvonen M.-R., Nevalainen, A Amoebae and other protozoa in material samples from moisture-damaged buildings. Environmental Research 96: II Yli-Pirilä T., Kusnetsov J., Hirvonen M.-R., Seuri M., Nevalainen A. Survival of amoebae on building materials Indoor Air 19: III Yli-Pirilä T., Kusnetsov J., Hirvonen M.-R., Seuri M., Nevalainen A Effects of amoebae on the growth of microbes isolated from moisture-damaged buildings. Canadian Journal of Microbiology 52: IV Yli-Pirilä T., Huttunen K., Nevalainen A., Seuri M., Hirvonen M.-R Effects of co-culture of amoebae with indoor microbes on their cytotoxic and proinflammatory potential. Environmental Toxicology 22: These articles are reproduced with the kind permission of their copyright holders. 12 1 INTRODUCTION More than 20 years of research has demonstrated that excess moisture and concurrent microbial growth in buildings is associated with adverse health effects suffered by the occupants (IOM 2004). Excess moisture can enter building structures in many ways, for example from leaks in the roof or plumbing, by capillary rise of ground moisture, or by condensation due to inadequate ventilation or thermal or water proofing. The excess moisture can cause damage and facilitate microbial growth in building structures, structural components or on the surfaces of the materials (Haverinen 2002). Different aspects of the exposure linked with moisture damage have been studied, such as microbial diversity and the presence of volatile organic compounds emitted by these microbes and moistened building materials, but no causative relationships between the experienced health problems and the exposure have been conclusively revealed so far (Bornehag et al. 2001). Studies on the microbes have concentrated on the fungi, and to some extent also on the bacteria present in moisture-damaged environments. Other organisms possibly present and possibly indicating moisturedamages have rarely been studied. However, it is likely that higher organisms able to consume fungi and bacteria as nutrition are also a part of the microbial network present at the moisture and mold damaged building materials. These higher organisms in this respect could include protozoa, such as amoebae, flagellates, ciliates, and even arachnids and insects (Flannigan 2001). Species of amoebae, flagellates and ciliates are ubiquitous in natural environments containing water. A gram of soil typically contains 10 4 to 10 5 cells of amoebae and flagellates (Ekelund and Rønn 1994) and a liter of natural water may be home to 10 5 to 10 6 protozoal cells (Zimmermann 1997). Because of their ubiquity, amoebae and other protozoa may also be transported into various man-made environments such as buildings. Possible routes of entry may include the remains of water or soil in soles of shoes, or via airborne route through doors, windows and other ventilation shafts. The availability of moisture determines whether amoebae can take up residence in a particular site inside a building. In contrast, the lack of food rarely prevents their growth, as these organisms can utilize a large variety of nutritional sources ranging from bacteria to algae. Amoebae require a water film to become active, that is to feed, move and replicate. The thickness of this water film depends on the size of the organism and can be as low as 5 μm. Therefore, some amoebae and other protozoa should be able to grow on moistened building materials. Many amoebae can survive even if the material dries out because many of the species are able to metamorphose 13 into resistant forms, cysts, in unfavorable environmental conditions like drought (Hausmann et al. 2003). The need to determine how common amoebae really are in moisture-damaged buildings arose from a case of a moisture-damaged hospital where several employees suffered serious adverse health symptoms. In the search of the cause for these severe symptoms, a thorough clinical testing was performed on the employees including the presence of IgG and IgA antibodies to Chlamydophila pneumoniae (Seuri et al. 2005). Surprisingly, 16 of total of 18 employees were positive for C. pneumoniae even though no clinical chlamydial infections had been observed. This phenomenon was thought to be possibly linked with the exposure to the conditions in the moisture-damaged building. However, C. pneumoniae and other Chlamydiarelated bacteria require a host organism and should not be able to survive as such on the moistened building materials. On the other hand, amoebae are natural hosts and carriers of Chlamydia-related bacteria (Amann et al. 1997; Birtles et al. 1997; Fritsche et al. 2000). It was considered possible that there were amoebae present in the moisture-damaged sites, and that the elevated antibody levels could be caused by Chlamydia-related bacteria residing inside of the amoebae (Seuri et al. 2005). Furthermore, over the years, amoebae had been occasionally detected in samples from buildings with suspected moisture-damage in routine cultivation for fungi and bacteria (unpublished observation). This led to a series of investigations focussed on amoebae in moisture-damaged buildings and building materials, the results of which are presented in this thesis. In order to elucidate the potential significance of amoebae in the exposure associated with moisture-damaged buildings, one necessary first step was to clarify the role of amoebae as members of the microbial network in the moisture-damaged environment. In this study, the occurrence of amoebae in moisture-damaged buildings is investigated, the ability of amoebae to grow on a selection of building materials is tested, and the effects of amoebae on the growth, viability, cytotoxicity, and proinflammatory potential of indoor bacteria and fungi are assessed. 14 2 REVIEW OF THE LITERATURE 2.1 Moisture-damage in buildings The exposing agents and health effects associated with moisturedamaged buildings The exposure in moisture-damaged buildings is a complex phenomenon in which both biological and chemical exposing agents may be released into the indoor air either in gaseous form or as attached to particles. The agents include spores and cells of microfungi, bacteria, yeasts, mites, protozoa, and their fragments; also toxins and other products of these organisms metabolism can be present in this multi-faceted exposure (Andersson et al. 1997; Glushakova et al. 2004; Hyvärinen et al. 2002; Nevalainen et al. 1991; Pasanen et al. 1992; Piecková and Wilkins 2004; Van Strien et al. 1994). Furthermore, moisture may also cause chemical reactions in the building materials resulting in release of volatile organic compounds (Korpi et al. 1998). Thus, quantifying the exposure is difficult, and the methods used today - such as measuring the concentration of viable microfungi in indoor air - can only be considered as surrogates of the actual exposure (Nevalainen and Seuri 2005). Nevertheless, the dampness-related exposure has been clearly shown to be associated with adverse health effects for those exposed (Bornehag et al. 2001; IOM 2004). It is also evident that the experienced health effects clearly differ from each other in different buildings with moisture damage, suggesting that the causes for the symptoms are probably not
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